Blood in a dish -----in vitro synthesis of red blood cells Presented by :Tian Jing Co-advisor: Dr.Ma and Dr.Jiang

Anemia 2 billion people worldwide and 10% of the US population, with the highest incidence among the elderly. Major surgery and trauma; A common toxicity of cancer therapies; 16 million red blood cell (RBC) transfusions every year in the United States. Background Source:

Need for RBC transfusions Obtained from donors Frequent supply bottlenecks Infectious risks ; Requires costly screening; Donors for rare blood types are scarce. Background Source:

Consequently, numerous efforts are underway to expand erythroid precursors and differentiate them in vitro into mature RBCs. Furthermore, erythroid precursors may ultimately serve as a novel cell-based therapy providing a renewable source of RBCs. Background

The first cell-based therapy The first successful blood transfusion: from one dog to another in 1665 In 1667, a sheep to man transfusion The first microscopic identification of RBCs by Antonie van Leeuwenhoek in The first successful human-to-human blood cell transfusion occurred with the treatment of postpartum hemorrhage using a husband-to-wife transfusion [1] [1]Diamond, L.K., McGraw-Hill Book Company(1980). Background Source:

The first cell-based therapy The first functional replacement therapy occurred in 1840 with whole blood transfusion treatment of hemophilia. The discovery of blood types by Karl Landsteiner in 1901 and earned him a Nobel Prize for Medicine in 1930 [2]. Background [2]Diamond, L.K., McGraw-Hill Book Company(1980). Source:

Erythropoiesis – the synthesis of RBCs Hematopoietic stem cells (HSCs); termed burst-forming units erythroid (BFU-E); colony-forming units erythroid (CFU-E); erythroid precursors termed proerythroblasts (ProE); basophilic erythroblasts (BasoE);polychromatophilic erythroblasts (PolyE) ; orthochromatic erythroblasts (OrthoE); reticulocytes (Retic) Introduction

In vitro production of RBCs This complex process of erythropoiesis, consisting of progressive phases : (1) Progenitor expansion; (2) Precursor amplification and maturation ; (3) Reticulocyte remodeling into terminal RBCs. Introduction

In vitro production of RBCs: the 2-step erythroid culture system Twenty years ago, Fibach [3] developed a liquid culture system that included two sequential steps: The first step contained glucocorticoids and conditioned media providing cytokines to promote erythroid ‘progenitor’ proliferation ; The second step contained EPO alone to promote survival of late- stage erythroid progenitor and maturation of erythroid precursors. [3] Fibach, E. Haematologia (1991). Introduction dexamethasone (Dex) ； erythro-myeloid progenitors (EMP)

Improvements of 2-step erythroid culture system The first step has been improved by the replacement of conditioned media with several defined cytokines [4] : SCF ; low concentrations of IL3 ; GM-CSF; EPO; To expand the number of BFU-E and maintain the survival of late-stage erythroid progenitors. [4]Malik, et al. Blood (1998). Introduction

Improvements of 2-step erythroid culture system It was also recognized that estradiol, as well as glucocorticoids, can inhibit erythroid maturation and lead to expanded numbers of erythroid ‘progenitors’ in the first phase of erythroid culture [5]. [9] Migliaccio, G. et al. Blood Cells Mol(2002). Introduction

Improvements of 2-step erythroid culture system The addition of insulin and thyroid hormone to EPO [6] ； Molecules antagonistic to the action of glucocorticoids and estrogens [7] ； DMSO, ferrous citrate and transferrin [8] ； Humanized serum proteins [9]. [6] Leberbauer, C. et al.. Blood (2005). [7] Miharada, K. et al.. Nat.Biotechnol (2006) [8] Maggakis-Keleman, C. et al. Biol. Eng. Comput(2003). [9] Migliaccio G. et al. Cell Transplant (2010). Introduction

Improvements of 2-step erythroid culture system The 2-step liquid cultures of human erythroid cells have traditionally generated less than 50% enucleated RBCs. Enucleation rates were dramatically improved by co- culture of erythroid precursors on a specific murine bone marrow (MS5) stromal cell line [10]. Efficient enucleation has also been facilitated using feeder-free conditions [11]. This is an important issue because the production of clinically useful RBCs in vitro will require strategies to avoid exposure of cellular products to nonhuman cells. [10] Giarratana, M.C. et al. Nat. Biotechnol (2005). [11] Miharada, K. et al. Nat. Biotechnol (2006). Introduction

Improvements of 2-step erythroid culture system Culture protocol for the efficient production of enucleated red blood cells without feeder cells from hematopoietic stem/progenitor cells. Passage I ∼ III are the steps to expand erythroid progenitor cells. Passage IV is the step to induce enucleation of progenitor cells [12]. MAP, mixture of D-mannitol, adenine, and disodium hydrogen phosphate dodecahydrate. nearly 80% of RBCs were enucleated [12] Miharada, K. et al. Nat.Biotechnol (2006). Introduction A B

Improvements of 2-step erythroid culture system Immature, multipotent hematopoietic progenitors have also been expanded in vitro by culture not only with cytokines but also by using human stromal cells transduced with hTERT [13] [13] Fujimi, A. et al. Int. J.Hematol (2008) hTERT: human telomerase catalytic subunit gene-transduced stromal cell Introduction

The recovery rate of RBC from the day 38 culture from filtration was 80.8 % 1.76 ×10 9 RBC were obtained from 500 CD34+ cells by the four-phase ‘‘stroma- supported macrophage co-culturing system’’ on day 38 Nearly 100% of the erythroblasts obtained from third-phase culturing with macrophages were enucleated in the medium both on day 36 and day 38

Ultimate goal Enucleated RBCs ; Oxygen delivery potential similar in vivo- generated RBCs ： Hemoglobin content, Oxygen dissociation characteristics, Membrane deformability, In vivo lifespan when injected into immunodeficient mice CD71, transferrin receptor; TER119, a cell surface antigen specific for mature erythroid cells. Summary

The problem of scale The RBC products require the ex vivo generation of cell numbers [14]; The costs associated with ex vivo erythroid cell expansion and differentiation; The tumorigenic potential [15] ; The establishment of an immortalized human erythroid cell line lacking the genes to produce A, B, and RhD antigens. [14] Giarratana, M.C., et al. Blood (2011). [15] H. Hentze,et al. Trends in Biotechnology, (2007). Conclusion

Thanks for attention!